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Methods for producing high density patterned cell arrays for biological assays

a cell array and high density technology, applied in the field of life sciences and biological processes, can solve the problems of difficult image analysis, difficult recognition of transfected cells from non-transfected cells, and complicated use and analysis of such “lawn-type” arrays

Inactive Publication Date: 2010-04-15
VALTION TEKNILLINEN TUTKIMUSKESKUS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a novel method for screening and molecular analysis of cells using "lawn-type" or "special coating" cell microarrays. The method involves layering cells over the array and allowing them to adhere for a specific time window, while controlling adhesion to pre-defined positions with empty space in between. The method also allows for the screening of test compounds and the analysis of cells based on their adhesion properties. The invention also provides a novel cell array comprising cells adhered only to the spots and a method for producing the array. The improvements of the invention enhance the analysis of cells and allow for the screening of drug targets essential for cancer, the screening of synthetic lethal effects of test compounds, and the comparison of functional response from one cell type to another. The invention also allows for co-culturing of different cell types and the use of untreated polystyrene as the background surface for the arrays, resulting in a significantly increased throughput compared to previous methods.

Problems solved by technology

This allows the cells to grow as a “lawn” all over the slides, and transfection can only be seen based on pre-defined coordinates, which makes image analysis difficult (see for example Fujimoto H et al.
Recognition of transfected cells from non-transfected is also very difficult as cells tend to migrate and mix when e.g. going through mitosis.
These issues complicate the use and analysis of such “lawn-type” arrays and can lead to false results.

Method used

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  • Methods for producing high density patterned cell arrays for biological assays
  • Methods for producing high density patterned cell arrays for biological assays
  • Methods for producing high density patterned cell arrays for biological assays

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cell Spot Microarrays for Genome Scale RNAi Analysis Using siRNA Reverse Transfection

I. Sample Preparation

Materials

[0091]384-Well Microarray Plates with cylindrical wells (Thermo Fisher Scientific Inc, cat. AB-1055)

Matrigel (BD Biosciences, Basement Membrane Matrix, Growth Factor Reduced (GFR), cat. 354230)

[0092]siRNA (Qiagen Human Druggable Genome siRNA Set V3.0)

siLentFect Transfection reagent (Bio-Rad Laboratories, cat. 170-3360)

Opti-MEM I Reduced-Serum Medium (Invitrogen, cat. 11058-021)

[0093]siRNA-matrix sample dilutions in 384 well format

[0094]Synthetic concetrated siRNA stocks were diluted with Opti-MEM to give final [siRNA]=1.67 μM*

[0095]5 μl of diluted siRNA was mixed with 0.5 μl of siLentFect transfection reagent** and incubated for 1 h at room temperature***

[0096]2 μl of Matrigel per siRNA sample was mixed with 2.5 μl of ice cold Opti-MEM and kept at +4° C.****

[0097]siRNA-lipid mixture and the diluted Matrigel were mixed together thoroughly to give final siRNA concentratio...

example 2

Co-Culture Cell Spot Microarrays for Genome Scale RNAi Analysis Using siRNA Reverse Transfection

[0125]Procedures I, II and IV have been carried out according to the corresponding procedures of Example 1.

III. Cell Application Procedure

Materials

[0126]Tissue Culture hood

VCaP human prostate cancer cells (ATCC, cat. CRL-2876)

RWPE-1 human prostate epithelial cells (ATCC, CRL-11609)

Foetal bovine serum (Gibco, cat. 10270106)

K-SFM Keratinocyte Serum Free Media (Invitrogen, cat. 17005-042)

[0127]HyQ®Tase™ (Thermo Fisher Scientific Inc, cat. SV30030.01)

CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate) (Invitrogen, cat. C2925)

CellTracker™ Red CMTPX (invitrogen, cat. C34552)

Cell Application

[0128]In a sterile cell culture laminar, 6×106 actively growing VCaP Human Prostate Cancer Cells* stained with cell permeant long term fluorescent cell tracing reagent CellTracker™ Green CMFDA****** cultured in K-SFM Keratinocyte Serum Free Medium, were washed once with PBS and detached from cell c...

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Abstract

The present invention relates to the fields of life sciences and biological processes. Specifically, the invention relates to microarrays and live cell based screening and molecular analysis. More specifically, the present invention relates to novel methods for the screening of the effects of a test compound on cells, for molecular analysis of the cells and for producing a microarray. The present invention also relates to cell arrays and the use of arrays for molecular analysis of the cells or for the screening of agents.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the fields of life sciences and biological processes. Specifically, the invention relates to microarrays and live cell based screening and molecular analysis. More specifically, the present invention relates to novel methods for the screening of the effects of a test compound on cells, for molecular analysis of the cells and for producing a microarray. The present invention also relates to cell arrays and the use of said arrays for molecular analysis of the cells or for the screening of agents. Furthermore, the present invention also relates to the use of said arrays to explore effects from one cell type to the other, to screen for drug targets essential for cancer, to screen for synthetic lethal effects of test compounds and / or to compare functional response from one cell type to another.BACKGROUND OF THE INVENTION[0002]Microarray technology has revolutionized research in the DNA, gene expression and protein function fiel...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B30/06C40B50/14C40B40/02
CPCG01N33/5032
Inventor RANTALA, JUHASAVIRANTA, PETRIKALLIONIEMI, OLLI
Owner VALTION TEKNILLINEN TUTKIMUSKESKUS